Revision as of 20:11, 24 September 2014 by >Mzcai
Agi26k0gap Probe Production
Production PCR
- Make Master Mix for 51 well (7 strips)
- 0 gap first round amplicon -> V6 primers
Components
|
1x rxn (ul)
|
50x rxn (ul)
|
First round amplicon (10nM) |
0.2 |
10
|
2x Kapa SYBR MM |
50 |
2500
|
100uM pAP1V6U |
0.4 |
20
|
100uM AP2V6 |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
|
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold
File:20140919 Agi26k0gapProductionPCR.PNG
EtOH Precipitation
- 6 5-ml tubes (with 8.5 wells of PCR product each) for 0gap
- 850ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 85ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 40ul and combined all probes into one 1.5ml
- Measured concentration with Nanodrop:
- ~225ul of Agi26k0gap probes: 335.3 ng/ul => 75.4 ug
Lambda Exonuclease Digestion
- Divide into eight pcr tubes of 29ul with total amplicon of ~10ug each
Components
|
Volume
|
Amplicon |
29
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
51
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 67.8ng/ul x 310ul = 21.0ug
Removal of Amplification Adapters
Components
|
Volume
|
ss-amplicon |
62
|
USER |
5
|
H2O |
13
|
total |
80
|
*~5ug ssDNA per tube
- Incubated at 37C for 2 hours
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
10
|
100uM RE-DpnII guide oligo |
5
|
RE-DpnII guide oligo:
0-gap: RE DpnII V6
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight
TBU Gel Check
- Combine the 5 tubes
- 2ul Sample + 2ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
- Digested sample
- Undigested sample: 2ul of Lambda digested but not USER/DpnII